AbstractBased on 16S rDNA sequence comparison, intracellular mycetome‐associated endosymbionts (P‐endosymbionts) of tsetse flies (Diptera: Glossinidae) form a distinct lineage within the γ‐3 subdivision of proteobacteria, related to the free‐living bacteriumEscherichia coli, midgut S‐endosymbionts of various insects including tsetse flies, and to the P‐endosymbiont lineage of aphids,Buchnera aphidicola.Gene organization and expression of several loci in intracellular microorganisms have revealed differences from free‐living bacteria. This study analyses two of these characteristics in tsetse endosymbionts; the copy number and gene organization of rDNA operons and the nature of the abundant protein(s) synthesized by these microorganisms. Results indicate thatGlossina morsitans morsitansS‐endosymbionts have multiple (seven) rDNA operons coding for 16S (rrs) followed by 23S (rrl) gene sequences, whereas tsetse P‐endosymbionts have a single, similarly organized rDNA operon. In tsetse mycetocytesin vitro, P‐endosymbionts synthesize a predominant protein of 60 kDa in size (p60) which by Western blot analysis shows immunological cross‐reactivity with the abundant 63 kDa (p63) protein ofB. aphidicola.p63 (also referred to as symbionin) has been characterized as a molecular chaperone, structurally and functionally similar to the groEL protein ofE. coli.Underin vitroconditions, tsetse S‐endosymbionts synthesize high levels of a similarly‐sized protein that cross‐reacts with p63 chaperonin. Antisera against the tsetse p60 protein also recognizes p63 protein ofB. aphidicola, suggesting that the abundant tsetse endosymb